Recombinant Human Active Raf-1 (Y340E Y341E, 306-end), CF

• Active Kinase - Active Raf-1(EE) (0.1 μg/μL) diluted with Kinase Dilution Buffer III and assayed as outlined in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
• Kinase Assay Buffer I - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerolphosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
• Kinase Dilution Buffer III - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with 50 ng/μL BSA solution.
• 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I. Store 200 μL aliquots at ≤ -20 °C.
• [³³P]-ATP Assay Cocktail - Prepare 250 μM [³³P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [³³P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I. Store 1.0 mL aliquots at ≤ -20 °C.
• Substrate - Inactive MEK1 and ERK1 were activated in a coupled reaction. Myelin Basic Protein (MBP) substrate diluted in distilled or deionized water to a final concentration of 1.0 mg/mL was subsequently used as a substrate for the activated ERK1.

1. Thaw the [³³P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
2. Thaw the Active Raf-1 (EE), Kinase Assay Buffer I, inactive ERK1, and inactive MEK1 on ice.
3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL:
   a. Diluted Active Raf-1(EE): 10 μL
   b. Inactive ERK1 (0.2 μg/mL): 1.0 μL
   c. Inactive MEK1 (0.2 μg/mL): 1.0 μL
   d. Kinase Dilution Buffer: 8.0 μL
4. Start the reaction with the addition of 5.0 μL ATP (250 μM) and incubate in a water bath at 30 °C for 15 minutes.
5. After the 15 minute incubation, remove 5.0 μL and add it to the following reaction components on ice, bringing the initial reaction volume up to 20 μL:
   a. Reaction Mixture: 5.0 μL
   b. MBP Substrate (1.0 mg/mL; on ice): 5.0 μL
   c. Distilled or deionized water (on ice): 10 μL
6. Set up the blank control as outlined in Step 5, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
7. Initiate the reaction with the addition of 5.0 μL [³³P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
8. After the 15 minute incubation, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
9. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
10. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
11. Determine the corrected cpm by removing the blank control value (see Step 6) for each sample and calculate the kinase specific activity as outlined below.
    
    
    Calculation of [³³P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5.0 μL [³³P]-ATP/pmol of ATP (in 5.0 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)
    
    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]

Scientific Data

SDS-PAGE View Larger

The approximate molecular weight is 63 kDa and the purity is > 85%.